Method of treating cardiovascular diseases

ABSTRACT

A method of reducing the negative effect of cadmium or aluminum ion or free-radicals in body tissues and a method of alleviating cardiovascular diseases and conditions by administering to a human or an animal a pharmaceutical composition comprising an amount of at least one specific isomer of inositol triphosphate sufficient to obtain said alleviation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of copending application Ser. No. 173,985, filed onMar. 28, 1988 which is now U.S. Pat. No. 4,019,566, which is acontinuation-in-part of U.S. patent application, Ser. No. 038,230 filedApr. 14, 1987 now abandoned which is a continuation-in-part of U.S.patent application, Ser. No. 015,679 filed Feb. 17, 1987, now U.S. Pat.No. 4,797,390, which is a continuation-in-part of U.S. patentapplication, Ser. No. 788,801 filed Oct. 18, 1985, now U.S. Pat. No.4,735,936.

FIELD OF THE INVENTION

The present invention relates to a method of preventing or alleviatingdifferent conditions in the body by administering thereto apharmaceutical composition comprising inositol triphosphate.

BACKGROUND OF THE INVENTION

Even as early as the year 1900, different researchers had reported thefinding of the organic phosphate compound phytic acid, i.e.,1,2,3,4,5,6-myo-inositol-hexakis(dihydrogenphosphate) (also sometimescalled inositolhexaphosphoric acid) in plants. The content of phyticacid in different plants varies considerably. The content in grain isusually approximately 0.5-2%, with certain exceptions. Polished rice hasa level of only 0.1% while wild rice contains as much as 2.2% phyticacid. Beans contain about 0.4-2%, oil plants approximately 2-5% andpollen 0.3-2%. The content of phytic acid in the plant varies during thegrowth period. The content is also influenced by, among other things,the climate.

In the literature there are reports of the presence of inositolpentaphosphate (IP₅) and inositol tetraphosphate (IP₄) in a few plants.It is further known that phosphate derivatives lower than IP₆ are formedat germination of grain. For instance, the final products at germinationare inositol and phosphate. The use of IP₆ has been described in severalscientific publications. The majority of the authors of these articleshave observed several negative effects on humans and animals whenconsuming IP₆ or substances containing IP₆. Feeding dogs with too highan amount of IP₆ gives rise for example to rachitis. In humans lack ofzinc and as a consequence thereof slower growth of children has beenobserved. Anemia has been observed mainly in women. Because of the abovementioned negative effects on the mineral balance in human and animals,attempts have so far been made to reduce the intake of IP₆ and itsderivatives to a minimum.

From C. A. Vol. 33 (1939), Abstr. No. 7351, No. 3/4 the use ofphosphates including inositol phosphates as an anti-rachitic diet hasbeen reported. No reference is made to specific inositol phosphates andnothing has been said in regard to complexing of metals.

U.S. Pat. No. 4,473,563 discloses the extra corporal treatment oferythrocytes to incorporate therein inositol phosphates to improve theoxygen supply. Then erythrocytes are separated from drawn blood whichhas been pumped out of the body for that purpose. After complicatedtreatment of erythrocytes the latter are re-introduced into the blood.There is no disclosure of administering inositol phosphates directly tothe body. Moreover, nothing has been said in regard to reduction of thenegative effect of cadmium or aluminum in the body or the inhibition orreduction of the formation of free-radicals or preventing or alleviatingdifferent conditions in the body by administering inositol phosphate.

In U.S. Pat. No. 2,723,938 the use of inositol phosphates is disclosedfor stabilizing dispersions of an aqueous suspension of penicillin. Thisensures that brief simple manual shaking will restore a state ofcomplete and uniform dispersion of the penicillin after prolongedstorage.

Cadmium has been found to be detrimental to human health and resultsfrom a large number of animal experiments obtained over many years shownegative effects even at very low levels of cadmium. This would meanthat a large proportion of the population is negatively affected, andthis is above all valid for smokers. Epidemiological research shows aconnection between the presence of cancer, high blood pressure andcardiovascular diseases (for instance, arteriosclerosis, heartinfarction, sudden cardiac death) and the occurence of cadmium in theenvironment. Exposure to cadmium also seems to be a factor in increasingthe risk of age diabetes.

In spite of very intensive research effort for many years seeking toprevent the above mentioned negative effects of cadmium and/or toprevent or alleviate the above mentioned problems created by cadmium,which in many cases involve very serious diseases, no good remedywithout side effects has till now been found.

Aluminum has recently been recognized as a health hazard. In dialysispatients, aluminum causes dementia and osteomalicia. It is suspectedthat aluminum may cause many abnormalities, such as Alzheimers diseasein humans. There are also investigations showing that aluminum can causeseveral diseases in animals. Aluminum can also increase lipidperoxidation in biological membranes, probably by destabilizing membranestructure. As for cadmium, no good remedy for Al-related diseases,without side effects has till now been found. Free-radicals have beensuggested to be involved in the pathology of a number of diseases, suchas autoimmune diseases and inflammatory diseases.

SUMMARY OF THE INVENTION

According to the present invention it has quite unexpectedly been foundpossible to reduce the above mentioned negative effects of cadmium,aluminum and free-radicals on humans and animals and thus to prevent oralleviate the connected diseases. Thus, a method of reducing thenegative effect of cadmium or aluminum ion or free-radicals in bodytissues has been brought about. At said method a pharmaceuticalcomposition comprising an amount of at least one specific isomer ofinositol triphosphate sufficient to interfere with cadmium or aluminumion or inhibit or reduce the formation of free-radicals in the body isadministered to a human or an animal.

In addition the present invention relates to a method of preventing oralleviating some diseases which may or may not be connected to cadmium,aluminum or free-radicals by administering to a human or an animal apharmaceutical composition comprising an amount of at least one specificisomer of inositol triphosphate sufficient to obtain said prevention oralleviation.

The invention also covers a method of facilitating a transplant and amethod of regulating unnormal levels of metal ions in a human or ananimal.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As examples of the diseases which the method according to the inventionis useful to prevent or alleviate, there are mentioned eye diseases suchas retinitis pigmentosa or damage of different parts of the eye such asthe lens, a lung disease such as bronchitis, emphysema or lung fibrosis,an inflammatory condition such as arthritis, high blood pressure,cardiovascular diseases such as artherosclerosis or increase of plateletaggregation, diabetes, cell proliferation changes, damage to the centralnervous system, a thrombotic condition such as damage to theendothelium, increase of platelet aggregation or inhibition of theprostacycline production, a hyperlipidaemic condition such as increasedcholesterol levels, a light induced disease such as skin damages. It isbelieved that at least some of the above diseases are caused oraggravated by the presence of cadmium, aluminum or free-radicals, butthe invention is not limited by said theory, since the method accordingto the invention is useful against said diseases whatever reason behindthem.

The method according to the invention is also intended to facilitate orimprove transplant of different organs and to regulate unnormal levelsof metal ions in the body.

For production of the isomer or isomers of IP₃ which accomplish theabove objectives and which is present in the composition used in themethod according to the invention, one or more of the compounds IP₆, IP₅or IP₄ or a natural product containing at least one of these compoundscan be used as a starting material. In the cases where the startingmaterial is a natural product, one with a content of at least 0.3%,preferably at least 1% of inositol phosphate (IP₆ +IP₅ +IP₄) ispreferably chosen. Particularly suitable products are beans, bran,pollen and oil plants.

The IP₃ isomers present in the composition used according to theinvention can, for example, be produced by enzymatic breakdown startingfrom IP₄, IP₅ and/or IP₆.

According to the invention a procedure where the above mentioned higherinositol phosphates IP₆, IP₅ and/or IP₄ are broken down enzymatically toIP₃ with phytase enzyme, for instance, is preferred. Phytase enzyme isnormally present in all inositol phosphate-containing plants and seeds.Because of this it is, according to the invention, usually not necessaryto add the enzyme if a natural product is used as starting material. Inthe cases where the natural product has too low an enzymatic activity orwhen IP₆, IP₅ or IP₄ or a mixture of these is used as starting material,a phytase enzyme, for example, from bran is added.

A suitable way to treat the natural or crude starting material is topretreat it, for instance by breakage or removal of outer membrane andremoval of unwanted constituents. Thus, when using pollen the allergensshould be removed. Thereafter, the material is soaked in water to makethe inositol phosphate available for breaking down and to activate theenzyme. In the cases where an extra quantity of enzymes is necessary,this quantity is added at this stage. The enzyme is then allowed to actfor so long a time as is necessary for the intended degree of hydrolysisto be achieved.

The hydrolysis takes place at a suitable temperature, usually 20°-70°C., preferably 30°-40° C. and at optimal pH-level for the phytasepresent. In order to stop the hydrolysis at the intended level theenzyme may be destroyed or inactivated, for instance by a rapid heatingof the hydrolysed starting material. This also ensures that anuncontrolled and undesired continued hydrolysis of IP₃ in the stomachwill not continue when the composition is administered. In order totransfer the material to a form which is stable at storage it cansuitably be freeze dried. Yeast can be used advantageously as a sourceof phytase. Preferably baker's yeast is used. When using yeastessentially only one isomer of IP₃ is obtained, namelyD-myo-inositol-1,2,6-triphosphate.

The above mentioned procedure, in applicable parts with possiblemodifications, can be used also when one or more of the compounds IP₆,IP₅ or IP₄ per se are used as starting material.

The pharmaceutical composition used in the method according to theinvention comprises as a pharmaceutically active ingredient at least onespecific isomer of inositol triphosphate (IP₃).

It is suitable that the composition used according to the inventionexists in unit dosage form. Tablets, granulates or capsules are suitableadministration forms for such unit dosage. Furthermore, tablets andgranulates can easily be surface treated such as to provide an entericcoating to prevent an uncontrolled hydrolysis in the stomach and tobring about a desired absorption in the intestine. Other suitableadministration forms are slow release and transdermal administration. Ausual pharmaceutically acceptable additive, excipient and/or carrier canbe included in the composition. The tablets or granulates can alsocontain a disintegrant which causes the tablets or the granulates,respectively, to disintegrate easily in the intestine. In certain cases,especially in acute situations, it is preferable to use the unit dosagein the form of a solution for intravenous administration.

The pharmaceutical composition can also consist as such of IP₃ solelywithout any additive, excipient or carrier.

If desired, the composition can be free of other inositol phosphatesIP₁, IP₂, IP₄, IP₅ and IP₆ Accordingly, the mixture of IP₃ isomers canhave a purity of 90-100%, such as 93-100% or preferably 95-100%.

Alternatively, the pharmaceutical composition used in the method canconsist of or comprise one or more specific IP₃ isomers disclosedhereinafter, each present in substantially pure form. Thus, thedifferent isomers can be isolated from each other in substantially pureform, which means that they have a purity of 80-100%, such as 82-100% or85-100%, preferably 90-100%. Since the isomers can be produced in pureform they can be mixed in any proportion, of course.

The production of IP₃ and the isolation of the different isomers thereofare disclosed in the co-pending U.S. patent application Ser. No.788,829.

It is in most cases suitable that the IP₃ -isomer or isomers in thecomposition used in the method according to the invention is present insalt form in order not to affect the mineral balance negatively. Thesalt should preferably consist of a medium, calcium, zinc or magnesiumsalt or a mixture of two or more of these salts. Calcium and zinc saltsor mixtures of these are especially preferred. The isomer of IP₃ canalso partly be present as a salt of one or more physiologicallyacceptable compounds in the lanthanide series.

For the above mentioned reasons it is also an advantage if thecomposition contains a surplus or an extra addition of at least onepharmaceutically acceptable salt of calcium, zinc, magnesium or copperwith a mineral acid or organic acid. This is especially valuable forolder persons who are often deficient in these minerals.

The composition used according to the present invention can preferablyalso contain at least one substance containing selenium, an unsaturatedfatty acid, such as gamma linoleic acid, vitamin E, vitamin C or apharmaceutically acceptable organic acid or salt thereof, such ascitrate, oxalate, malonate and tartrate. These substances also help tocounteract the negative effect of cadmium, aluminum and the formation offree-radicals in the body and/or to give in addition thereto, in certaincases, a desirable effect together with the IP₃ isomer in thecomposition. The content of selenium in the composition is preferablysuch that the daily intake is about 0.7-8 ug/kg body weight preferably0.7-3.3 ug. For vitamin E the corresponding values are about 0.1-2 mgand 0.1-1 mg, respectively.

The composition is suitably free from penicillin.

For administration to human patients appropriate dosages can routinelybe determined by those skilled in this art by extension of the resultsobtained in animals at various dosages. The preferred dosage for humansfalls within the range of 0.1 to 100, especially 0.1-50 mg IP₃ /day/kgbody weight.

In animal experiments, no toxic effects were seen after administrationof very high doses of IP₃, 160 mg/kg body weight by intravenousinjection to mice or 1600 mg/kg body weight by intraperitoneal injectionto mice.

The composition used according to the present invention contains atleast one, sometimes two or more of the following substances, whichcorrespond to the essential IP₃ -isomer or isomers mentioned above:

D-myo-inositol-1,2,6-triphosphate of the formula ##STR1## where X ishydrogen, at least one univalent, divalent or multivalent cation, or amixture thereof, n is the number of ions, and z is the charge of therespectively ion;

D-myo-inositol-l,2,5-triphosphate of the formula ##STR2## where X, n andz have the above mentioned meaning;

myo-inositol-1,2,3.-triphosphate of the formula ##STR3## where X, n andz have the above mentioned meaning;

L-myo-inositol-1,3,4-triphosphate of the formula ##STR4## where X, n andz have the above mentioned meaning.

In each of the above formulas n ranges between 6 to 1 inclusive and zranges from 1 to 6 inclusive. Preferably, n is between 3 to 6 inclusiveand z is 3, 2 or 1. Of above isomers D-myo-inositol-1,2,6-triphosphateis preferred.

Other inositol triphosphate isomers that may be utilized in the presentinvention as the active IP₃ ingredient in the composition have thestructural formula ##STR5##

One group of inositol triphosphate compounds is defined by structuralformula (I) where three of R₁, R₃, R₅, R₇, R₁₀ and R₁₁ are hydroxyl andthe remaining three are phosphate and R₂, R₄, R₆, R₈, R₉ and R₁₂ arehydrogen.

Another group of inositol triphosphates is defined by structural formula(I) where three of R₁, R₃, R₆, R₇, R₉ and R₁₂ are hydroxyl and theremaining three are phosphate and R₂, R₄, R₅, R₈, R₁₀ and R₁ l arehydrogen.

Still another group of inositol triphosphates is defined by structuralformula (I) where three of R₁, R₃, R₅, R₈, R₁₀ and R₁₂ are hydroxyl andthe remaining three are phosphate and R₂, R₄, R₆, R₇, R₉ and R₁ l arehydrogen.

Yet another group of inositol triphosphates is defined by structuralformula (I) where three of R₁, R₄, R₅, R₈, R₉ and R₁₂ are hydroxyl andthe remaining three are phosphate and R₂, R₃, R₆, R₇, R₁₀ and R₁ l arehydrogen.

Still yet another group of inositol triphosphates is defined bystructural formula (I) where three of R₁, R₃, R₆, R₈, R₉ and R₁₂ arehydroxyl and the remaining three are phosphate and R₂, R₄, R₅, R₇, R₁₀and R₁ l are hydrogen.

Even still another group of inositol triphosphates is defined bystructural formula (I) where three of R₁, R₃, R₆, R₇, R₁₀ and R₁₂ arehydroxyl and the remaining three are phosphate and R₂, R₄, R₅, R₈, R₉and R₁ l are hydrogen.

Even yet group of inositol triphosphates is defined by structuralformula (I) where three of R₁, R₃, R₅, R₈, R₁₀ and R₁₁ are hydroxyl andthe remaining three are phosphate and R₂, R₄, R₆, R₇, R₉ and R₁₂ arehydrogen.

Finally, another group of inositol triphosphates is defined bystructural formula (I) where three of R₁, R₃, R₅, R₇, R₉ and R₁₁ arehydroxyl and the remaining three are phosphate and R₂, R₄, R₆, R₈, R₁₀and R₁₂ are hydrogen.

Particular inositol triphosphate compounds within the contemplation ofthe above formula include compounds having the structural formula (I)where

R₅, R₇ and R₁₀ are phosphate, R₁, R₃ and R₁ l are hydroxyl and R₂, R₄,R₆, R₈, R₉ and R₁₂ are hydrogen;

R₁, R₁₀ and R₁ l are phosphate, R₃, R₅ and R₇ are hydroxyl and R₂, R₄,R₆, R₈, R₉ and R₁₂ are hydrogen;

R₁, R₃ and R₁ l are phosphate, R₅, R₇ and R₁₀ are hydroxyl and R₂, R₄,R₆, R₈, R₉ and R₁₂ are hydrogen;

R₃, R₅ and R₇ are phosphate, R₁, R₁₀ and R₁ l are hydroxyl and R₂, R₄,R₆, R₈, R₉ and R₁₂ are hydrogen;

R₃, R₇ and R₁₀ are phosphate, R₁, R₅ and R₁ l are hydroxyl and R₂, R₄,R₆, R₈, R₉ and R₁₂ are hydrogen;

R₃, R₁₀ and R₁₁ are phosphate, R₁, R₅ and R₇ are hydroxyl and R₂, R₄,R₆, R₈, R₉ and R₁₂ are hydrogen;

R₁, R₃ and R₆ are phosphate, R₇, R₉ and R₁₂ are hydroxyl and R₂, R₄, R₅,R₈, R₁₀ and R₁ l are hydrogen;

R₆, R₇ and R₉ are phosphate, R₁, R₃ and R₁₂ are hydroxyl and R₂, R₄, R₅,R₈, R₁₀ and R₁₁ are hydrogen;

R₃, R₅ and R₈ are phosphate, R₁, R₁₀ and R₁₂ are hydroxyl and R₂, R₄,R₆, R₇, R₉ and R₁₁ are hydrogen;

R₁, R₃ and R₁₂ are phosphate, R₅, R₈ and R₁₀ are hydroxyl and R₂, R₄,R₆, R₇, R₉ and R₁₁ are hydrogen;

R₁, R₃ and R₅ are phosphate, R₈, R₁₀ and R₁₂ are hydroxyl and R₂, R₄,R₆, R₇, R₉ and R₁₁ are hydrogen;

R₁, R₅ and R₈ are phosphate, R₃, R₁₀ and R₁₂ are hydroxyl and R₂, R₄,R₆, R₇, R₉ and R₁₁ are hydrogen;

R₁, R₅ and R₁₂ are phosphate, R₃, R₈ and R₁₀ are hydroxyl and R₂, R₄,R₆, R₇, R₉ and R₁₁ are hydrogen;

R₁, R₃ and R₁₂ are phosphate, R₆, R₈ and R₉ are hydroxyl and R₂, R₄, R₅,R₇, R₁₀ and R₁₁ are hydrogen;

R₁, R₃ and R₆ are phosphate, R₇, R₁₀ and R₁₂ are hydroxyl and R₂, R₄,R₅, R₈, R₉ and R₁₁ are hydrogen;

R₄, R₅ and R₈ are phosphate, R₁, R₉ and R₁₂ are hydroxyl and R₂, R₃, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₃, R₅ and R₈ are phosphate, R₁, R₁₀ and R₁₁ are hydroxyl and R₂, R₄,R₆, R₇, R₉ and R₁₂ are hydrogen;

R₁, R₃ and R₅ are phosphate, R₈, R₁₀ and R₁₁ are hydroxyl and R₂, R₄,R₆, R₇, R₉ and R₁₂ are hydrogen;

R₁, R₃ and R₅ are phosphate, R₇, R₉ and R₁₁ are hydroxyl and R₂, R₄, R₆,R₈, R₁₀ and R₁₂ are hydrogen;

R₁, R₃ and R₁₂ are phosphate, R₅, R₈ and R₉ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₁, R₃ and R₈ are phosphate, R₅, R₉ and R₁₂ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₃, R₅ and R₁₂ are phosphate, R₁, R₈ and R₉ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₁, R₅ and R₉ are phosphate, R₃, R₈ and R₁₂ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₁, R₅ and R₁₂ are phosphate, R₃, R₈ and R₉ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₁, R₃ and R₉ are phosphate, R₅, R₈ and R₁₂ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₅, R₉ and R₁₂ are phosphate, R₁, R₃ and R₈ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₁, R₈ and R₉ are phosphate, R₃, R₅ and R₁₂ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₁, R₈ and R₁₂ are phosphate, R₃, R₅ and R₉ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₅, R₈ and R₁₂ are phosphate, R₁, R₃ and R₉ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₁, R₉ and R₁₂ are phosphate, R₃, R₅ and R₈ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₅, R₈ and R₉ are phosphate, R₁, R₃ and R₁₂ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₃, R₈ and R₉ are phosphate, R₁, R₅ and R₁₂ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₃, R₉ and R₁₂ are phosphate, R₁, R₅ and R₈ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen;

R₃, R₈ and R₁₂ are phosphate, R₁, R₅ and R₉ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen; and

R₈, R₉ and R₁₂ are phosphate, R₁, R₃ and R₅ are hydroxyl and R₂, R₄, R₆,R₇, R₁₀ and R₁₁ are hydrogen.

The above formula describes specific isomers of inositol triphosphatewhere the inositol is selected rom the group myoinositol, cisinositol,epiinositol, alloinositol, neoinositol, mucoinositol, chiroinositol andscylloinositol.

IP₃ may be the sole pharmaceutically active ingredient in thecomposition used. However, also other pharmaceutically activeingredients can be present therein. The amount of IP₃ should thenconstitute 5 to 95 or 15 to 80, such as 25 to 60 per cent by weight ofsaid active ingredients.

Moreover, the composition can be a multi-vitamin unit containing 2 to60, such as 2 to 40 or preferably 2 to 25 per cent by weight of IP₃based on the total weight of pharmaceutically active ingredients.

The composition usually contains 0.01-1.5 g, such as 0.05-1.3 orpreferably 0.1-1 g of IP₃.

The invention also comprises a method of preventing or alleviating oneof the following conditions; damage to cell membranes, damage to theplacenta, the prostate or the testicles, damage to the conducting systemof the heart, migraine headache, menstruation disorders, kidney damage,an allergy or multiple sclerosis, and wherein said condition may or maynot be attributable to, caused or aggravated by the presence of cadmium,aluminum or free-radicals in the body.

The method comprises administering to a human or an animal apharmaceutical composition comprising an amount of at least one specificisomer of inositol triphosphate sufficient to obtain said prevention oralleviation.

Furthermore, the invention covers a method of alleviating thedetrimental effect of radiation in the body, which method comprisesadministering to a human or an animal an amount of radiation. Forinstance, the radiation can be X-ray or nuclear radiation, but otherkinds of radiation are also contemplated.

The invention is further explained below in connection with embodimentexamples of which example 1 and 2 show that platelet aggregation can becounteracted by injection or oral administration of IP₃. Example 3relates to the effect of IP₃ in changing the distribution of cadmium indifferent organs of mice. Example 4 shows that IP₃ prevents an increaseof platelet aggregation in humans caused by smoking. In example 5 it isshown that an increased blood glucose level in mice can be counteractedby injection of IP₃. Examples 6 and 7 show that IP₃ prevents or reducesthe formation of free-radicals. Example 8 illustrates experiments onbinding constants between different metals and IP₃. Examples 9 and 10teach production of IP₃. Example 11 shows the production of a solutionof a potassium salt of IP₃ for injection and example 12 shows theproduction of tablets of the calcium salt of IP₃. Example 13 shows thereversal of platelet aggregation by addition of IP₃. In example 14 it isshown how to reduce the formation of thrombus. Examples 15 and 16illustrate the reduction of hypertension by addition of IP₃. Example 17relates to the beneficial effect of IP₃ on lipid levels. In example 18IP₃ is shown to reduce the occurence of artherosclerosis. Example 19describes the reduction of inflammation by addition of IP₃. Example 20teaches the protective effect of IP₃ against damage on transplantedorgans. The iron binding effect of IP₃ is illustrated in example 21.Examples 22-25 show the production of different inositol triphosphatecompounds.

METHOD FOR EXAMPLES 1 AND 2

Rabbits (New Zealand white, males) weighing 2-2.5 kg were used. Theywere fed a diet free from inositol phosphates, for 10 days before theexperiment.

ANIMAL EXPERIMENTAL PROCEDURE

In example 1 (intravenous injection of test-substances), the followingprocedure was used:

    ______________________________________                                        Time:      Treatment                                                          ______________________________________                                        0 minutes: Intravenous injection of inositol phosphate                                   in 1 ml physiological saline, or 1 ml                                         physiological saline respectively.                                 1 minute:  Blood sample 1 (9 ml + 1 ml 3.8% sodium                                       citrate) taken.                                                    2 minutes: Intravenous injection of 4 microgram Cd as                                    CdCl.sub.2 in 0.5 ml physiological saline, or 0.5                             ml physiological saline respectively.                              5 minutes: Blood sample 2 (9 ml + 1 ml 3.8% sodium                                       citrate) taken.                                                    ______________________________________                                    

In example 2 (oral administration of test substances) the same procedurewas used except that the first injection was replaced by oraladministration of the inositol phosphates or saline respectively.Injection volumes were the same as those above. The oral dosing was made1 hour before blood sample 1. The blood sampling and the secondintravenous injection were made as above. The rabbits wereunanesthetized during the experiments.

TREATMENT OF SAMPLES

The two blood samples from each animal were centrifuged at 1200revolutions per minute, for 10 minutes, and the plasma with plateletswas obtained.

The plasma with platelets from the two samples was analyzed concerningthe response to addition of ADP (adenosin diphosphate) in anaggregometer (Chronopar Corp Mod, 440) according to Born (J. Physiol:67,1968). The two samples were analyzed simultaneously at the sameconcentration (1-20 micromolar) of ADP, in the two channels of theinstrument.

The principle of this test is that the plasma with platelets is turbid,and has a low transmittance for light. As ADP is added, the plateletsaggregate and form clumps. This results in an increase of transmittancewhich is quantified by the instrument. The response to ADP was measuredin scale units, with 80 scale units representing maximal aggregation. Inorder to have a maximal sensitivity of the method to pick up changes inplatelet reactivity, the ADP dose should cause a response of 5-30 scaleunits. This was normally achieved with 5 uM ADP, but in some animals alower or higher does (1-20 uM) was necessary.

The result of the test is expressed as maximal aggregation in sample 2(scale units) minus maximal aggregation in sample 1.

An increase in platelet aggregation has been reported to occur aftersmoking, and in cardiovascular diseases, and agents which suppressplatelet aggregation are believed to be of value in treating, forexample, cardiovascular diseases.

EXAMPLE 1

Methods were as above. Four different isomers of IP₃ were tested, theintravenously injected amount was 2×10⁻⁷ mol. The isomers wereD-myo-inositol-1,2,6-triphosphate (1,2,6),L-myo-inositol-1,3,4-triphoshate (1,3,4),myo-inositol-1,2,3-triphosphate (1,2,3) andD-myo-inositol-1,2,5-triphosphate (1,2,5).

The results were as follows:

    ______________________________________                                                                    Change in aggregation                                                         from sample 1 to                                  Injection 1                                                                             Injection 2 No    sample 2 (scale units)                            ______________________________________                                        1,2,6     Cd          15    -0.4                                              1,2,3     Cd          12    +0.3                                              1,3,4     Cd          13    +0.6                                              1,2,5     Cd          14    +1.7                                              Saline    Cd          14    +3.4                                              ______________________________________                                    

The results show that all the tested isomers had a good effect inpreventing the cadmium-induced aggregation, and that the best effect wasobtained with 1,2,6.

EXAMPLE 2

The methods were as above. D-myo-inositol-1,2,6-triphosphate (IP₃), dose2×10⁻⁵ mol, was given orally.

The following results were obtained:

    ______________________________________                                                                    Change in aggregation                             Oral                        from sample 1 to                                  administration                                                                           Injection  No    sample 2                                          ______________________________________                                        Saline     Cd         23    +1.9                                              IP.sub.3   Cd         15    -0.4                                              ______________________________________                                    

At the does used i this experiment, IP₃ prevented the effect of Cd onplatelet aggregation.

EXAMPLE 3

Mice weighing 18-20 gram at the start of the experiment were used.During the experiment and for at least seven days before the experimentthe mice were fed a semisynthetic diet free of inositol phosphates. Themice were divided in two groups.

They received daily intraperitoneal injections of physiological salineor D-myo-inositol-1,2,6-triphosphate (IP₃) for 9 days. The dose of IP₃was 10⁻⁶ mol/day and the injected volume was 0.2 ml.

On day two of the experiment, 5-10 minutes after the secondintraperitoneal injection, all mice received an intravenous injection of2.5 microcurie of ¹⁰⁹ Cd as cadmium chloride in 50 ul of saline. Afterthe last intraperitoneal injection the mice were killed and severalorgans were dissected out and weighed.

Radioactivity in the different organs was measured by counting with agamma-counter. Radioactivity in the organs of the IP₃ -treated animalwas compared with that of control animals, which had been treated withsaline for the same period of time. In the results radioactivity in theorgans of the animals treated with IP₃ is expressed as % of theradioactivity found in controls. The results were as follows:

    ______________________________________                                        Organ       Cd-level compared to control (%)                                  ______________________________________                                        Lung        74                                                                Heart       67                                                                Aorta       65                                                                Spleen      57                                                                Salivary gland                                                                            87                                                                Liver       100                                                               Kidney      104                                                               ______________________________________                                    

These results show that I₃ causes a strong decrease in cadmiumconcentration in lung, aortic artery, heart and spleen. Liver and kidneylevels were not affected.

EXAMPLE 4

The effect of D-myo-inositol-1,2,6-triphosphate (IP₃) on plateletaggregation after smoking in humans was studied.

Four young healthy male non-smokers received, on two occasions, acapsule containing 50 mg of IP₃ or 50 mg of a placebo. Neither subjectnor investigator knew whether the subject had received IP₃ or placebo.

Two hours after ingestion of the capsule, a blood sample was obtained.The subject then smoked two cigarettes in rapid succession. A secondblood sample was obtained after smoking. The aggregation responses ofthe platelets to ADP and collagen in the two samples were determined,using essentially the same procedure as in example 1. The results areexpressed as change in aggregation from the pre-smoking to thepost-smoking sample. A positive sign indicates that aggregation wasstronger after smoking.

    ______________________________________                                                 Concentration              Difference                                Aggregating                                                                            of aggregating             between IP.sub.3                          agent    agent       IP.sub.3                                                                              Placebo                                                                              and placebo                               ______________________________________                                        ADP      0.5    mmol     +1.5  +7.25  5.85                                    "        1      mmol     -1.5  +0.25  1.75                                    "        2.5    mmol     -1.5  0      1.5                                     "        5      mmol     -2.5  -0.75  1.75                                    Collagen 0.5    mg       +5.15 +12.25 6.5                                     "        1      mg       -8.25 +1.75  10.0                                    "        2.5             -3.75 0      3.75                                    "        5      mg       -1.5  -0.25  1.25                                    ______________________________________                                    

In the placebo group, smoking caused an increase in aggregation, whichwas most marked at low concentrations of aggregation agents. In allcases this effect was counteracted by IP₃ Thus, IP₃ prevents increase ofplatelet aggregation caused by smoking.

EXAMPLE 5

Mice, 10 in each group, were injected intraperitoneally with the Na-saltof D-myo-inositol-1,2,6-triphosphate (IP₃) in three dose levels or withphysiological saline. 30 minutes after this injection, all mice exceptone control group received an intravenous injection of alloxan, 50 mg/kgin saline.

The animals were starved for 12 hours before, and one hour after thealloxan injection. 72 hours after the alloxan injection, a blood samplefrom the mice was analyzed with respect to glucose level. The resultswere as follows:

    ______________________________________                                        Dose of IP.sub.3                                                                           Dose of alloxan                                                  mg/kg        mg/kg       Blood glucose                                        ______________________________________                                        0             0          216                                                  0            50          864                                                  800          50          857                                                  1600         50          677                                                  ______________________________________                                    

Alloxan causes diabetes and increased blood glucose level by promotionfree-radical reactions in the insulin producing cells. With IP₃ therewas a dose-dependent decrease in blood glucose levels, and the highestdose gave some protection to the alloxan.

EXAMPLE 6

Lipid peroxidation was studied in lipid micelles. The following reactionmixture was incubated for 2 hours at 37° C.:

    ______________________________________                                        0.4 ml         Clark- Lubs buffer pH 5.5                                      0.2 ml         phospholipid liposomes                                         0.1 ml         IP.sub.3 0.5-5 mM or 0.1 ml H.sub.2 O                          0.1 ml         Fe.sup.2+ 1 mM or 0.1 ml H.sub.2 O                             0.1 ml         Al.sup.3+ 4 mM or 0.1 ml H.sub.2 O                             0.1 ml         H.sub.2 O                                                      ______________________________________                                    

The IP₃ was D-myo-inositol-1,2,6-triphosphate. After incubation, 0.5 mlof Thiobarbituric acid+0.5 ml 25% HCl was added and the mixture washeated at 100° C. for 15 minutes. 1 ml Lubrol PX 1% (Sigma) was addedand lipid peroxidation was measured by measuring absorbance at 532 nm.The results were as follows:

    ______________________________________                                                 Concentration, mM                                                                            Absorbance                                            Experiment Fe.sup.2+                                                                            Al.sup.3+ IP.sub.3                                                                            532 nm                                      ______________________________________                                        1          0.1    0         0     0.36                                        2          0      0.4       0     0.12                                        3          0.1    0.4       0     0.89                                        4          0.1    0.4       0.5   0.36                                        5          0.1    0         0.5   0.30                                        6          0.1    0         0.4   0.26                                        7          0.1    0         0.2   0.29                                        8          0.1    0         0.1   0.28                                        9          0.1    0         0.05  0.27                                        10         0      0         0     0.13                                        ______________________________________                                    

Fe²⁺ caused lipid peroxidation (group 1 vs 10). Al³⁺ itself caused noperoxidation (2 vs 10) whereas the combination of Fe²⁺ +Al³⁺ caused muchstronger peroxidation than Fe²⁺ alone (1 vs 3). Addition of IP₃completely prevented the interaction between Fe²⁺ and Al³⁺ (3 vs 4). Insystems with only Fe²⁺, IP₃ caused marked reduction in radical formation(1 vs 5-9).

EXAMPLE 7

Reaction mixtures with the following compositions were incubated for 5minutes at 37° C.:

    ______________________________________                                        KH.sub.2 PO.sub.4 buffer pH 7.4                                                                   20 mM                                                     EDTA                 0.1 mM                                                   Salicylate           1 mM                                                     Ascorbate            1 mM                                                     H.sub.2 O.sub.2      3.3 mM                                                   Fe.sup.3+            0.05 mM                                                  IP.sub.3             0, 2.5, 5 or 10 mM                                       ______________________________________                                    

The products formed by oxidation of salicylate were quantified withHPLC. The IP₃ was D-myo-inositol-1,2,6-triphosphate.

The system studies radical scavenging. Under these reaction conditions,all Fe³⁺ will form complex with EDTA. The Fe-EDTA complex will inducefree-radical formation, and the ability of IP₃ to prevent oxidation ofsalicylate is studied.

The results of the experiment were:

    ______________________________________                                        Concentration Relative amount of                                              of IP.sub.3, mM                                                                             salicylate oxidized                                             ______________________________________                                        0             100                                                             2.5           44                                                              5             43                                                              10            19                                                              ______________________________________                                    

Thus, IP₃ is able to act as a radical scavenger, thereby preventingfree-radical induced damage to other molecules or tissues.

EXAMPLE 8

The relative metal binding constants forD-myo-inositol-1,2,6-triphosphate (IP₃) and calcium, cadmium, aluminumand iron respectively were determined.

A solution consisting of 4 mM IP₃ was titrated with 100 mM NaOH. Similarconcentrations were performed in the presence of Ca, Cd (12 mM) and Al,Fe (8 mM).

A strong metal complex will result in a lowering of pH at a certainamount NaOH added. The performed titrations show the relative metalbinding properties. At pH 8 the binding properties are as follows:Ca<Cd<Fe<Al.

EXAMPLE 9 Hydrolysis of sodium phytate with baker's yeast andfractionation of a mixture of inositol phosphates

A 0.7 gram quantity of sodium phytate (from corn, Sigma Chemical Co) wasdissolved in 600 ml sodium acetate buffer pH 4.6. 50 gram of baker'syeast from Jastbolaget, Sweden (dry substance: 28%, nitrogen content:2%, phosphorus content: 0.4%) was added with stirring and incubation wascontinued at 45° C. The dephosphorylation was followed by determiningthe inorganic phosphorus released. After 7 hours when 50% inorganicphosphorus was liberated the hydrolysis was stopped by adding 30 ml ofammonia to pH 12. The suspension was centrifuged and the supernatant wascollected.

400 ml of the supernatant was passed through an ion-exchange column(Dowex 1, chloride form, 25 mm×250 mm) and eluted with a linear gradientof hydrochloric acid (0-0.7 N HCl). Aliquots of eluted fractions werecompletely hydrolyzed in order to determine the contents of phosphorusand inositol. The peaks correspond to different inositol phosphates i.e.a peak with the ratio of phosphorus to inositol of three to one consistsof inositol triphosphates etc.

EXAMPLE 10 Structural determination of isomers of inositoltriphosphate..

The fraction obtained in example 9 with a phosphorus/inositol ratio ofthree to one was neutralized and evaporated before analysis with H-NMR.Data show that the peak consists of myo-inositol-1,2,6-triphosphate.

EXAMPLE 11 Solution of potassium salt ofD-myo-inositol-1,2,6-triphosphate for injection

0.5 g of the potassium salt of IP₃ and 0.77 g NaCl were dissolved in98.73 ml of water for injection to form a solution suitable forinjection into a person or an animal.

EXAMPLE 12 Tablets of calcium salt of D-myo-inositol-1,2,6 triphosphate

Tablets of the calcium salt of D-myo-inositol-1,2,6-triphosphate wereproduced in the following way. 50 g calcium salt ofD-myo-inositol-1,2,6-triphosphate, 132 g lactose and 6 g acacia weremixed. Purified water was then added to the mixture, whereupon themixing was continued until a suitable consistency was obtained. Themixture was sieved and dried. Then the mixture was blended with 10 gtalcum and 2 g magnesium stearate. The mixture was compressed intotablets each weighing 200 mg.

EXAMPLE 13

Two groups, each consisting of six rats, were fed a casein-based dietcontaining approximately 30% saturated fat. One group receivedD-myo-inositol-1,2,6-triphosphate (IP₃), 200 ppm, in the diet and theother group served as a control.

Platelet aggregation in response to thrombin was measured after twomonths.

The results were as follows:

    ______________________________________                                                 Thrombin induced platelet                                            Treatment                                                                              aggregation (cm) Number of animals                                   ______________________________________                                        Control  2.1 ± 0.9     6                                                   IP.sub.3 0.5 ± 0.01    6                                                   ______________________________________                                    

Thus, after two months of supplementation of IP₃ thrombin inducedaggregation was significantly lower in the IP₃ -treated animals comparedto the controls.

EXAMPLE 14

Thrombus formation induced by ADP in the microcirculation of the hamstercheek pouch was determined in the presence ofD-myo-inositol-1,2,6-triphosphate (IP₃) compared to control.

The cheek pouch is everted in the anaesthetized animal and amicropipette containing ADP is placed close to a venule. ADP moves intothe vessel and induces the formation of a thrombus on the wall of thevenule. The growth rate of the thrombus is used to evaluate the effectof anti-thrombotic compounds.

IP₃, 5 mg/kg, was administered by infusion during a 10-minute period.Thrombus growth rates were determined at an interval of 5 minutes. Tenminutes after infusion 40% inhibition in growth rates was obtained,which shows IP₃ to be an efficient anti-thrombotic compound.

EXAMPLE 15

Chronic administration of low oral doses of cadmium to rats inducesmoderate hypertension.

64 female rats (4 groups of 16 rats) were fed a rye-based Cd-free diet.The experiment was divided into two phases:

Phase 1: Induction of hypertension; groups 1 and 2 served as controls;groups 3 and 4 were given 0.1 ppM Cd in the drinking water.

Phase 2: Reversal of Cd-induced hypertension; no group received Cd;groups 2 and 4 received 60 ppm D-myo-inositol-1,2,6-triphosphate (IP₃)in the drinking water.

The systolic blood pressure was measured by the tail cuff method at 4week intervals throughout the experiment and the following results wereobtained:

    ______________________________________                                                  Systolic blood pressure (mm Hg)                                               Phase 1   Phase 2                                                   ______________________________________                                        Group 1     120         120                                                   Group 2     119         125                                                   Group 3     136         138                                                   Group 4     133         124                                                   ______________________________________                                    

The results show that Cd-induced hypertension can be reversed by addingIP₃ (group 4).

EXAMPLE 16

Chronic administration of low oral doses of lead to rats inducesmoderate hypertension.

45 female rats (3 groups of 15 rats) were fed a rye-based Pb-free diet.The experiment was divided into two phases:

Phase 1: Induction of hypertension; group 1 served as control; groups 2and 3 were given 1 ppm Pb in the drinking water.

Phase 2: Reversal of Pb-induced hypertension; group 3 received 200 ppmD-myo-inositol-1,2,6-triphosphate (IP₃) in the diet.

The systolic blood pressure was measured by the tail cuff method at 4weeks intevls throughout the experiment and the following results wereobtained:

    ______________________________________                                                  Systolic blood pressure (mm Hg)                                               Phase 1   Phase 2                                                   ______________________________________                                        Group 1     122         120                                                   Group 2     133         134                                                   Group 3     132         121                                                   ______________________________________                                    

The results show that Pb-induced hypertension can be reversed by addingIP₃ (group 3).

EXAMPLE 17

Thirty male rats were fed a standard diet supplemented with 2%cholesterol and 1% cholic acid for a 14-day pretreatment period. Duringthe subsequent 14-day treatment period one group of 10 rats was giventhe above diet with 0.2% D-myo-inositol-1,2,6-triphosphate (IP₃) added,while the other rats continued on the pretreatment diet and served as acontrol. The effect on serum concentration of lipid- and cholesterolmetabolites is summarized below (%):

    ______________________________________                                                Total     HDL       Triglyc-                                                                             β-lipo-                                                                        Total                                Treatment                                                                             cholesterol                                                                             cholesterol                                                                             erides protein                                                                             lipids                               ______________________________________                                        Control 100       100       100    100   100                                  IP.sub.3                                                                               78       116        98     82    95                                  ______________________________________                                    

Thus, it can be seen that the levels of cholesterol and β-lipoprotein isdecreased, while the level of HDL-cholesterol is increased in theanimals treated with IP₃. These data show that IP₃ has a beneficialeffect on the lipids in the body.

EXAMPLE 18

Fourteen rabbits were fed with a standard diet supplemented with 0.05%cholesterol and 18% butter. Six animals were also given 200 mg/kgD-myo-inositol-1,2,6-triphosphate (IP₃) added to the diet. Theexperiment was continued for 6 months. After this period the animalswere killed and the severity of artherosclerosis in the aorta wasdetermined. The results showed that in 4 out of 6 IP₃ treated animals,there were no lesions, while all 8 animals in the control group wereartherosclerotic.

Thus, the occurence and severity of the artherosclerosis wasdramatically reduced in the IP₃ -treated animals.

EXAMPLE 19

Injection of carrageenan into the subplanar surface of the rat hind-pawinduces an inflammatory response that results in pronounced oedema. Thedegree of oedema can be reproducibly quantified by measuring pawcircumference.

Two groups of five male rats were injected 2 hours and 1 hour beforeinjection of carrageenan with 1000 mg/kgD-myo-inositol-1,2,6-triphosphate (IP₃) or Krebs ringer solutionrespectively.

Measurement of the paw diameter was made at hourly intervals.

The results show that an injection of IP₃ reduces the inflammation with40% four hours after the induction of the inflammation. Thus, theinflammation in the IP₃ -treated animals was strongly reduced.

EXAMPLE 20

Transplant of an organ from one animal to another includes first areduction of blood flow followed by reflow of blood in the new animal.During this process the organ is easily damaged and as a measurement theso called Schiff's base is determined.

In one group of six kidneys from rabbits the blood flow was firstreduced for 120 minutes followed by reflow for 60 minutes. The othergroup of six kidneys was processed in the same way but two doses of 50ppm D-myo-inositol-1,2,6-triphosphate (IP₃) were administered 5 minutesbefore flow reduction and 5 minutes before reflow. Schiff's base wasdetermined in cortex and medulla of the kidneys (% of control):

    ______________________________________                                                      Schiff's base                                                   Treatment       Cortex  Medulla                                               ______________________________________                                        Control         100     100                                                   IP.sub.3         27      65                                                   ______________________________________                                    

Thus, IP₃ -treated kidneys are strongly protected against damage duringtransplant.

EXAMPLE 21

In an in vitro system consisting of cultured erythroleukemic cells (cellline K562) the ironchelating properties ofD-myo-inositol-1,2,6-triphosphate (IP₃) were studied.

The cells are preincubated with radio labelled iron.

The cells are immersed into an IP₃ -containing aqueous solution at atemperature of 37° C. The amount of iron released to the solution wasmeasured. The results show that four times as much iron was releasedwhen IP₃ was present in the solution. This shows that IP₃ binds ironstrongly.

EXAMPLE 22

A 0.5 gram quantity of D-chiro-inositol was dissolved in 1 ml phosphoricacid at 60° C. 20 g polyphosphoric acid was added and the mixture washeated to 150° C. under vacuum for 6 yours. The mixture was diluted withwater to a volume of 200 ml and passed through an ion-exchange column(Dowex 1, chloride form, 25 mm×250 mm) and eluted with a linear gradientof hydrochloric acid (0-2.0 N HCl).

The content of the peak with the ration of phosphorus to inositol of sixto one was precipitated by addition of calciumhydroxide. The precipitatewas filtered, washed and mixed with 10 ml of a cation-exchange resin togive the acid form of the inositolhexaphosphate. After neutralizationwith sodium hydroxide and freeze-drying the sodium salt ofD-chiro-inositolhexaphosphate was obtained.

EXAMPLE 23

A 0.8 gram quantity of epi-inositol was dissolved in 1.5 ml ofphosphoric acid at 60° C. 32 g polyphosphoric acid was added and themixture was heated to 150° C. under vacuum for 6 hours. The mixture wasdiluted with water to a volume of 200 ml and passed through anion-exchange column (Dowex, chloride form, 25 mm×250 mm) and eluted witha linear gradient of hydrochloric acid (0-2.0 N HCl).

The content of the peak with the ratio of phosphorus to inositol of sixto one was precipitated by addition of calcium hydroxide. Theprecipitate was filtered, washed and mixed with 10 ml of acation-exchange resin to give the acid form of theinositolhexaphosphate. After neutralization with sodium hydroxide andfreeze-drying the sodium salt of epi-inositolhexaphosphate was obtained.

EXAMPLE 24

A 0.8 gram quantity of the sodium salt of D-chiro-inositolhexaphosphateproduced according to Example 22 was dissolved in 300 ml sodium acetatebuffer, pH 5.2. 1.3 gram wheat phytase (EC 3.1.3.26, 0.015 U/mg fromSigma Chemical Co.) was added and the mixture was incubated at 38° C.

After the liberation of 50% inorganic phosphorus the hydrolysis wasstopped by adding ammonia to pH 12.

The mixture containing D-chiro-inositolphosphates was passed through anion-exchange column (Dowex 1 chloride form, 25 mm×250 mm) and elutedwith a linear gradient of hydrochloric acid (0-0.7 N HCl).

The peak with the ratio of phosphorus to inositol of three to one wasneutralized with 1.0 M sodium hydroxide and freeze-dried.

Structural determination with NMR and IR showed the product to beD-chiro-inositoltriphosphate.

EXAMPLE 25

A 1.2 gram quantity of the sodium salt of epi-inositolhexaphosphateproduced according to Example 23 was dissolved in 500 ml sodium acetatebuffer, pH 5.2. 2.0 gram wheat phytase (EC 3.1.3.26, 0.015 U/mg fromSigma Chemical Co.) was incubated at 38° C.

After the liberation of 50% inorganic phosphorus the hydrolysis wasstopped by adding ammonia to pH 12.

The mixture containing epi-inositolphosphates was passed through anion-exchange column (Dowex 1, chloride form, 35 mm×250 mm) and elutedwith a linear gradient of hydrochloric acid (0-0.7 N HCl).

The peak with the ratio of phosphorus to inositol of three to one wasneutralized with 1.0 M sodium hydroxide and freeze-dried.

Structural determination with NMR and IR showed the product to beepi-inositoltriphosphate.

For purposes of further understanding the invention, formulas are givenbelow of the IP₃ isomers of the invention. Formulas are also given forIP₆, IP₅, IP₄ and IP₂.

The lower phosphate-esters of myoinositol are named depending on wherethe phosphoric acid groups are situated on the inositol ring, with thenumbering giving as low position numbers as possible. L and D stand forclock-wise and counterclock-wise counting respectively, and are useddepending on which result gives the lowest position number. The carbonatom which has an axial phosphoric acid group always has the positionnumber 2. The structural formulas below are simplified to the acid form.

    ______________________________________                                         ##STR6##       myo-inositol; C.sub.6 H.sub.6 (OH).sub.6                       ##STR7##                                                                      ##STR8##       1,2,3,4,5,6-hexakis-(dihydrogen- phosphate)-myo-inositol                      alternatively myo-inositol hexakis (phosphate) or                             IP.sub.6                                                       ##STR9##       D-myo-inositol-1,2,6-triphosphate alternatively                               D-1,2,6-IP.sub.3                                               ##STR10##      D-myo-inositol-1,2,5-triphosphate alternatively                               D-1,2,5-IP.sub.3                                               ##STR11##      myo-inositol-1,2,3-triphosphate alternatively                                 1,2,3-IP.sub.3                                                 ##STR12##      L-myo-inositol-1,2-diphosphate alternatively                                  L-1,2-IP.sub.2                                                 ##STR13##      D-myo-inositol-1,2,5,6-tetra- phosphate or                                    D-1,2,5,6-IP.sub.4                                             ##STR14##      L-myo-inositol-1,2,3,4,5-penta phosphate or                                   L-1,2,3,4,5-IP.sub.5                                          ______________________________________                                         P = OPO.sub.3 H.sub.2                                                    

I claim:
 1. A method of alleviating cardiovascular disease comprisingadministering to a human or an animal in need thereof a cardiovasculardisease alleviating effective amount of a pharmaceutical compositionwhich comprises at least one specific isomer of inositol triphosphate.2. A method according to claim 1, wherein the cardiovascular disease isartheroschlerosis or increase of platelet aggregation.
 3. A methodaccording to claim 1 wherein the pharmaceutical composition comprises atleast one isomer of inositol triphosphate selected from the groupconsisting of D-myo-inositol- 1,2,6-triphosphate,d-myo-inositol-1,2,5-triphosphate, myo-inositol-1,2,3-triphosphate andL-myo-inositol-1,3,4-triphosphate.
 4. A method according to claim 2wherein the pharmaceutical composition comprises at least one isomer ofinositol triphosphate selected from the group consisting ofD-myo-inositol-1,2,6-triphosphate, D-myo-inositol-1,2,5-triphosphate,myo-inositol-1,2,3-triphosphate and L-myo-inositol-1,3,4-triphosphate.